Delivery of therapeutic agent

ABSTRACT

A method of producing nanovesicles comprising an oligonucleotide inhibitor to an oncogene or a proto-oncogene or the gene product thereof, said method comprises a) introducing a DNA sequence encoding an oligonucleotide capable of inhibiting a human oncogenic or proto-oncogenic transcription factor, into a mammalian cell; b) allowing the cell to express said inhibitor oligonucleotide; and c) obtaining nanovesicles containing said inhibitor oligonucleotide from said cell. Nanovesicles produced by the claimed method can be effectively and specifically targeted to e.g. cancer cells to deliver the inhibitor oligonucleotide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/EP2013/073740, filed Nov. 13, 2013, which is hereby incorporated inits entirety by reference and which claims the benefit of Swedishapplication no. 1251290-1, filed on Nov. 13, 2012, also incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to the delivery of potentially therapeuticagents via cell-derived vesicles.

BACKGROUND OF THE INVENTION

Cancer is a leading cause of death worldwide. In 2008, various cancersaccounted for 7.6 million deaths (around 13% of all deaths), accordingto the World Health Organization (WHO), and this number is projected tocontinue rising, with an estimated 13.1 million deaths worldwide in 2030[GLOBOCAN 2008 (IARC) Section of Cancer Information (Nov. 11, 2012)].

Malignant melanoma is responsible for a majority of deaths caused byskin tumors, and is the most common malignancy in young adults. Whenmelanoma is metastatic, prognosis is unfavorable, and the therapeuticalternatives are few. Uveal melanoma has different clinical featuresthan skin melanomas, and often spreads primarily to liver. For uvealmelanoma it has been proposed that approximately one out of two patientswill develop metastases within 15 years after treatment of the primaryocular tumor. The average survival time after diagnosis of livermetastases is 8 to 10 months and the mortality rate in patients withliver metastases of uveal melanoma is 92% over two years. One treatmentoption is regional perfusion of the liver with hyperthermia andmelphalan. This approach is however very invasive and associated withrisks of surgical complications. Even though remissions are seen in mostcases, survival is only marginally prolonged.

Melanoma of the skin and uveal melanoma are merely two examples ofcancer, which takes the lives of millions of people each year. Treatmentof cancer is therefore one of the major challenges facing modernmedicine.

In general, current anti-cancer therapeutic strategies include surgery,radiotherapy, cytotoxic drugs (chemotherapy) and hormone drugs. Adrawback of chemotherapeutic agents is that they are unselective andcause adverse side effects, which effectively limits dosage and hencealso the therapeutic effect. There is a need for improved, moreselective cancer therapies.

A rare example of a medication directly targeted to a point mutationoncogene is vemurafenib, targeting specifically the BRAF^(V600E)mutation common in skin melanomas, with relatively less off-targeteffects compared to chemotherapy. However this treatment only prolongslife with a few months in metastatic melanoma, and only works in thosemelanomas that have this specific mutation. A possibility wouldtherefore be to target the downstream signaling molecules. Thedownstream targets however lack enzymatic activity, and are oftenimpossible to treat with small molecules, because of their tertiarystructure. Further, as a small molecule inhibitor would distributethroughout the body, it would also cause systemic side effects innormal, healthy cells that are depending on such downstream molecules.

Over the last decade, gene therapy has received increased attention andit is believed that in the future gene therapy may be applicable to manydiseases, including cancer.

The most common form of gene therapy involves using DNA that encodes afunctional, therapeutic gene in order to replace a mutated gene. Othertypes of gene therapy involve directly correcting a mutation, or usingDNA that encodes a therapeutic protein drug (rather than a natural humangene) to provide treatment. It has also been suggested that RNAinterference, referred to as RNAi, may be possible to exploit in genetherapy against e.g. infections or cancer.

RNAi is a gene regulating mechanism occurring in many eukaryotic cellswhich has been known since the late 1990's, and which has been, usedprimarily as a research tool to achieve gene knockout.

However, a major challenge for the development of RNAi-basedtherapeutics and other gene therapy applications is delivery of thetherapeutic agent (e.g. an RNA molecule). Viral vectors may beefficient, but give rise to safety concerns, and can be rapidlyeliminated by a patient's immune system, reducing its impact.

The mode of delivery is particularly important when the target is a geneor gene product that is important to the function also of normal healthycells. Therefore, delivering a drug in a concentrated way to a diseasedcell specifically has the potential to reduce systemic side effects andside effects in other cells.

Hence, one obstacle to successful targeting of genes and proteinsinvolved in disease, including cancer, is the development of a safe andeffective method of delivering the therapeutic agent to the target.

SUMMARY OF THE INVENTION

It is an object of the invention to at least partly overcome thedrawbacks of the existing technologies, and to provide a means ofdelivering a therapeutic agent, in particular an oligonucleotide, totarget cells.

In a first aspect, these and other objects are achieved by a method ofproducing nanovesicles comprising an inhibitor oligonucleotide to anoncogene or a proto-oncogene or the gene product thereof, said methodcomprising

a) introducing a DNA sequence encoding a nucleic acid capable ofinhibiting a human oncogene or proto-oncogene or a gene product thereof,into a mammalian cell;

b) maintaining the cell under conditions allowing expression of saidoligonucleotide inhibitor; and

c) obtaining nanovesicles containing said oligonucleotide inhibitor fromsaid cell.

The step of obtaining the nanovesicles may include isolation naturallyreleased extracellular vesicles from the cell culture, and/or producingnanovesicles by extrusion of the cell through micro and/or nanofilters.

As used herein, “oncogene” refers to a gene that has the potential tocause cancer. In cancer cells, oncogenes are often mutated or expressedat high levels (upregulated or amplified). A “proto-oncogene” refers toa gene that can become an oncogene due to mutations or increasedexpression.

Examples of known proto-oncogenes include RAS, WNT, MYC, ERK, and TRK.

As used herein, “nanovesicle” refers to a cell-derived vesicle having asize in the nanometer range, and typically less than 500 nm.“Cell-derived vesicles” include extracellular vesicles or exosomesnaturally released from cells, as well as vesicles produced from cellsby serial extrusion of the cells through micro and/or nano filters, asdescribed herein. Nanovesicles produced from cells by serial extrusionmay be referred to as “artificial nanovesicles”, however it should benoted that “artificial” in this context only means that the nanovesiclesare not naturally released by the cells (as opposed to, for example,exosomes). Hence, “artificial” in this context does not suggest that thenanovesicles are synthetic; they are still cell-derived and areconstituted of the same components as the cell from which thenanovesicle originated (“producer cell”).

As used herein, “inhibit” means that the expression of a gene resultingin a functional gene product (e.g. a protein) is reduced, e.g. byinhibition of transcription, mRNA degradation, or inhibition of mRNAtranslation into protein. In the context of the present invention, the“inhibition” need not be complete; partial inhibition or repression ordown-regulation may also provide a desirable effect.

As used herein, “oligonucleotide inhibitor” refers to an oligonucleotidewhich is an inhibitor, i.e. is capable of inhibiting a target molecule.It may also be referred to as an “oligonucleotide inhibitor”.

The method present invention is advantageous, in particular compared tothe use of synthetic delivery vehicles such as synthetic liposomes,since the production of nanovesicles can easily be scaled up byculturing more of the nanovesicle-producing cells, and the nanovesiclescould be produced in large quantities at a suitable time-point, e.g.when the cell is expressing optimal levels of targeting molecule.Additionally, the nanovesicles obtained by the present invention, inparticular the nanovesicles obtained by serial extrusion of the producercells, may contain higher concentrations of the inhibitoroligonucleotide (higher loading capacity), and may be easier to loadwith an inhibitor oligonucleotide such as siRNA. Furthermore, variousmodification of the nanovesicles, e.g. by expression of targetingmolecules on the surface, is enabled or at least greatly simplified. Themethod of the invention is also cost-effective, since ananovesicle-producing cell culture can be scaled up by normal cellculturing, and also frozen, which reduces waste associated withshelf-life of the cells.

In embodiments of the invention the inhibitor oligonucleotide is aninhibitor to a human oncogenic or proto-oncogenic transcription factor.By targeting a transcription factor, many different malignancies couldbe repressed by one inhibitor. Preferably, the inhibitor oligonucleotideis may be an inhibitor to a human MYC gene or gene product, preferablythe c-Myc gene or gene product, or an inhibitor to a member of the E2Fgene family or a gene product thereof. In other embodiments, theinhibitor oligonucleotide may be an inhibitor to a human cellular signaltransduction molecule lacking enzymatic activity, for example a memberof the Ras family.

In embodiments of the invention, the oligonucleotide may be RNA,preferably an RNAi molecule. An RNAi molecule may be selected from thegroup consisting of shRNA, siRNA, miRNA, piRNA, nasiRNA or antisenseRNA, or any other molecule with RNAi capacity.

In embodiments of the invention, the DNA sequence encoding anoligonucleotide capable of inhibiting a human oncogene or proto-oncogenemay be a DNA sequence selected from the group consisting of SEQ ID NO:1-92 and SEQ ID NO: 185-218, preferably selected from the groupconsisting of SEQ ID NO: 1-32, SEQ ID NO: 33-67, and SEQ ID NO: 185-218,more preferably selected from among SEQ ID NO: 1-10 and SEQ ID NO:185-196.

In embodiments of the invention, the cell may express a targetingmolecule on the cell surface.

As used herein, “targeting molecule” means a molecule capable ofspecifically binding to another molecule (“target molecule”). Hence, atargeting molecule may be used to specifically localize e.g. a cell or avesicle presenting the targeting molecule on its surface to an entity,such as a cancer cell, presenting the target molecule.

The targeting molecule may be a ligand specifically binding to a surfaceprotein that is overexpressed on cancer cells. Expression of targetingsurface molecules in the nanovesicle-producing cells may providetissue-specific targeting of the nanovesicles obtained from the cells.An advantage with making the producer cell express the targetingmolecule is that the cell creates a membrane bound molecule that may bemore efficient in interacting with targeting receptors, whereas theefficacy of liposome-engineered targeting often can fail.

Optionally, the method may comprise introducing a DNA sequence codingfor a targeting molecule to be expressed on the cell surface, prior tostep a) or simultaneously with step a).

In embodiments of the invention, the cell may express at least twodifferent targeting molecules, which may further increase targetingspecificity or otherwise improve the targeting of the cells and/or thenanovesicles to cancer cells.

Examples of suitable targeting molecules include epidermal growth factor(EGF), melanocyte-stimulating hormone (MSH) or a variable region of anantibody directed against a surface protein that is overexpressed oncancer cells, or a fab-fragment of said antibody.

In embodiments of the invention, the cell is a human cell and theoncogene or protooncogene is a human oncogene or proto-oncogene. In suchembodiments, the human cell may be transfected or transduced with agenetic construct comprising a gene whose gene product functionallyreplaces the human oncogene or proto-oncogene naturally expressed by thehuman cell. For example, the human oncogene or proto-oncogene may be onemember of the MYC gene family, and the genetic construct may compriseanother member of the MYC gene family.

In embodiment of the invention, the cell may be transfected ortransduced with a construct comprising the N-Myc or L-Myc genes andoverexpresses N-Myc or L-Myc.

In embodiments of the invention, step a) of the above method maycomprise introducing a genetic construct comprising said DNA sequencecoding for an oligonucleotide capable of inhibiting human c-Myc,operatively linked to an inducible promoter, and step b) may compriseinducing expression by activation of said inducible promoter.

The method of producing nanovesicles according to the invention may beperformed in in vitro or ex vivo.

In another aspect, the invention provides an isolated nanovesicleproduced by the method described above.

As used herein, “isolated nanovesicle” refers to a nanovesicle that hasat least been separated from the producer cell, typically separated fromthe producer cell culture or from a first filtrate obtained whenproducing artificial nanovesicles by serial extrusion through micro- ornanofilters as described below. Isolated nanovesicles are typicallycontained in a medium or composition, and may have been subject to oneor more isolation or purification steps to remove e.g. cell debris orother components originating from cells, for example by centrifugationand/or filtration.

Although the present specification recites “a nanovesicle”, it isunderstood that this term also encompasses a plurality of nanovesicles.

In a further aspect, the invention provides an isolated nanovesiclecomprising an inhibitor oligonucleotide to a human oncogene orproto-oncogene, preferably a human oncogenic or proto-oncogenictranscription factor. In particular, the oncogene or proto-oncogene maybe a member of the MYC gene family, and more preferably the geneencoding human c-Myc. Alternatively the oncogene or proto-oncogene maybe a member of the E2F gene family, and more preferably a human geneencoding human E2f1, E2f2 or E2f3. As another example of, the oncogeneor proto-oncogene may be a member of the RAS gene family, and morepreferably a human gene encoding human H-Ras, N-Ras or K-Ras.

In embodiments of the invention the inhibitor oligonucleotide may beRNA, preferably an RNAi molecule. The RNAi molecule may be selected fromthe group consisting of shRNA, siRNA, miRNA, piRNA. nasiRNA or antisenseRNA, or any other molecule with RNAi capacity. In some embodiments, theRNA may be shRNA or siRNA comprising a sequence selected from the groupconsisting of SEQ ID NO: 93-184, preferably selected from the groupconsisting of SEQ ID NO: 93-124 (targeting myc) and 125-159 (targetingE2f), for example selected from among SEQ ID NO: 93-102 (targetingc-myc).

However the oligonucleotide may also be DNA, e.g. an antisenseoligonucleotide (ASO).

In a further aspect, the present invention provides a human celltransfected or transduced with a genetic construct comprising a genewhose gene product functionally replaces a human oncogene orproto-oncogene naturally expressed by said human cell. Hence, theproducer cell may be modified to be independent on the normal,non-pathological expression of a gene intended to be inhibited by theinhibitor oligonucleotide. As a result, the growth and viability of theproducer cell can be unaffected by the production of inhibitoroligonucleotide. In particular, the human oncogene or proto-oncogene maybe one member of the MYC gene family, and the genetic constructintroduced into the cells may be another member of the MYC gene family.For example, the human oncogene or proto-oncogene may be c-Myc and thegenetic construct may comprise a gene coding for N-Myc or L-Myc.

In embodiments of the invention, the human cell may further comprise aDNA sequence coding for a inhibitor oligonucleotide to said oncogene orproto-oncogene.

In a further aspect, the invention provides a human cell transduced ortransfected with a DNA sequence coding for an inhibitor oligonucleotideto a human oncogene or proto-oncogene, preferably a human oncogenic orproto-oncogenic transcription factor, wherein said DNA sequence isoperably linked to an inducible promoter.

A human cell as comprising a DNA sequence coding for an inhibitoroligonucleotide to a human oncogene or proto-oncogene may be comprise aDNA sequence selected from the group consisting of SEQ ID NO: 1-92 andSEQ ID NO: 185-218, preferably selected from the group consisting of SEQID NO: 1-32, SEQ ID NO: 33-67, and SEQ ID NO: 185-218, more preferablyselected from among SEQ ID NO: 1-9 and SEQ ID NO: 185-196.

In embodiments of the invention, the human cell may be a stem cell,preferably an embryonic stem cell or a mesenchymal stem cell.

In another aspect, the invention provides a non-human mammalian cell,transduced or transfected with a DNA sequence coding for an inhibitoroligonucleotide to a human oncogene or proto-oncogene.

The invention may be used for delivery of a therapeutic agent to a cell,in vitro or in vivo, e.g. for therapy. Hence, in another aspect theinvention provides a method of delivering a inhibitor oligonucleotideagainst a human oncogene or proto-oncogene to a cell, comprisingcontacting said cell with a human cell or a nanovesicle as describedabove.

In embodiments, a method of delivering a inhibitor oligonucleotideagainst a human oncogene or proto-oncogene to a cancer cell in vivo,comprises the steps of

a) administering to a patient having said cancer cell, a cell transducedor transfected with a DNA sequence coding for an inhibitoroligonucleotide to a human oncogene or proto-oncogene, wherein said DNAsequence is operably linked to an inducible promoter, and

b) inducing expression of the inhibitor oligonucleotide byadministration of a substance activating said inducible promoter.

Hence, nanovesicles as described herein may be used for treatment ofcancer.

In yet another aspect, the invention relates to a composition comprisingnanovesicles as described herein and a pharmaceutically acceptablecarrier. The composition and/or the nanovesicles may be for use as amedicament, in particular for treatment of cancer. The cancer to betreated may be selected from the group consisting of malignant melanoma,colorectal cancer, ovarian cancer, breast cancer, renal cancer,gastrointestinal cancer, brain cancer, lung cancer, nasopharyngealcancer, esophageal cancer, gastric cancer, liver cancer, cervicalcancer, prostate cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, nasalNK/T-cell lymphoma, sarcoma, leukemia, neuroendocrine cancers, midlinecarcinomas, neuroblastoma and cancers of the head and neck.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the realization that nanovesiclesoriginating from cells, including mammalian cells, can be used asvehicles for the delivery of potentially therapeutic inhibitor moleculesthat target fundamental cellular functions, with increased efficacy anddecreased risk for adverse side effects, compared to prior art deliverytechnologies.

In particular, the present inventors propose the use of cells and/orvesicles as described herein for delivery of nucleic acids that mayselectively inhibit or repress the production of, or functionality of,an oncogene or proto-oncogene or a corresponding gene product, forexample oncogenic or proto-oncogenic transcription factors and/oroncogenic or proto-oncogenic signaling molecules. The oncogene orproto-oncogene may be a non-mutated oncogene.

One family of oncogenes or proto-oncogenes of particular interest is theMYC gene family consisting of MYC, MYCN and MYCL. The MYC gene familyencodes three different transcription factors, c-Myc (encoded by MYC),N-Myc (encoded by MYCN) and L-Myc (encoded by MYCL), respectively, whichregulate a wide array of genes and cellular processes that are crucialin malignant disease. However, the MYC gene family is also active innormal cell proliferation.

The gene encoding c-Myc, MYC, is located on chromosome 8 in the humangenome. The Myc protein acts though binding of Enhancer Box sequences(E-boxes) and also by recruiting histone acetyltransferases (HATs), thusregulating the chomatin structure. Myc is believed to regulateexpression of 15% of all human genes.

c-Myc and N-Myc are downstream of mitogenic signals such as Wnt, Shh andEGF (via the MAPK/ERK pathway). Since these pathways are often activatedby mutation of genes like APC (Wnt-pathway), PATCHED (Shh-pathway) andEGFR, BRAF, RAS, PTEN (EGF-pathway), c-Myc or N-Myc is induced in cancercells.

MYC can also be activated by chromosomal translocation to atranscriptionally active chromosomal location, such as those of theImmunoglobulin locus in B lymphocytes and of the T-cell receptor in Tlymphocytes. These translocation result in various hematologicalmalignancies such as Burkitt lymphoma or T-acute lymphocytic leukemia.

An alternative activation of MYC family members is via amplification,which happens in various cancers including lung cancer (MYC, MYCN andMYCL), neuroblastoma (MYCN) and breast cancer (MYC).

By modifying expression of other genes, Myc activation results innumerous biological effects. The first to be discovered was itscapability to stimulate metabolism; e.g. it stimulates the transcriptionof genes encoding ornithine decarboxylase and lactate dehydrogenase. Mycalso drives cell proliferation (e.g. by upregulating cyclins, E2ftranscription factors and downregulating p21), and it plays a veryimportant role in regulating cell growth (upregulating ribosomal RNA andproteins), apoptosis (downregulating Bcl-2), differentiation and stemcell self-renewal.

Targeting the most common of the Myc proteins, c-Myc, with smallmolecules has been proven exceedingly difficult because the tertiaryprotein structure lacks pockets that could function as binding sites forsmall inhibitory molecules. However, it has been shown that theexpression of Myc proteins can be blocked by RNAi molecules (Wang, H.,et al., c-Myc depletion inhibits proliferation of human tumor cells atvarious stages of the cell cycle. Oncogene, 2008. 27(13): p. 1905-15).

One set of genes that are regulated downstream of Myc and other cellsignaling pathways is the transcriptional activators of the E2F family.E2F1, E2F2 and E2F3A encode transcription factors that stimulate genesinvolved in cell cycle progression such as cyclin E and Cdk2, componentsof DNA replication (for example DNA polymerase, replicationorigin-binding protein HsOrc1, MCM 5 and cdc6) and nucleotide biogenesis(thymidine kinase and dihydrofolate reductase). The E2F family alsocontains transcriptional repressors (E2f3b and E2f4-8).

E2f activators are bound by the retinoblastoma protein pRB, encoded bythe RB1 gene. When bound, E2f proteins are unable to stimulate cellcycle progression. To stimulate cell cycle progression, pRB isphosphorylated by cyclin-dependent kinases Cdk2, Cdk4 and Cdk6 onmultiple sites. This releases bound E2f from pRB resulting in E2factivation. Cancer cells can exhibit elevated E2f activities because ofmany reasons including loss of the RB1 gene or the CDKN2A-C genes. Thelatter encode Cdk inhibitors p15, p16 and p18, collectively called Ink4proteins, which inhibit Cdk4 and Cdk6. Other ways to activateconstitutive E2f activity is by direct gene transcription of CDK4, CCND2and even E2F1 genes by Myc proteins, or by Ink4-resistant mutations ofCdk4.

Targeting the most common of the E2f proteins, E2f1, with smallmolecules is difficult since it is a transcription factor and hence lackan enzymatic active site. However, it has been shown that the expressionof E2f1 can be blocked by RNAi molecules in melanoma, resulting in cellcycle arrest and senescence. (Verhaegen et al. E2F1-Dependent OncogenicAddiction of Melanoma Cells to MDM2 Oncogene. 2012 Feb. 16;31(7)828-841)

In embodiments of the invention, signaling molecules targeted by theinhibitor molecule may be signaling molecules lacking enzymaticactivity. The signaling molecules targeted by the inhibitor molecule mayinclude signaling molecules involved in intracellular signaltransduction, e.g. regulating cell growth, differentiation and survival,such as the Ras family of proteins, including Kras, Hras, and Nras.

According to embodiments the present invention, nanovesicles containingan inhibitor molecule can be produced in vitro, from animal cells grownin vitro, typically mammalian cells such as murine or human cells. Thecells from which nanovesicles are produced are herein referred to as“producer cells”. In embodiment of the invention, the nanovesicles maycomprise exosomes and other extracellular vesicles naturally releasedfrom cells, as well as artificial vesicles produced by serial extrusionsof cells through micro- and/or nano-filters. In other embodiments of theinvention the nanovesicles are artificial nanovesicles, which may beproduced by serial extrusions of cells through micro- and/ornano-filters. It is known that extruding cells through filtersdisintegrates the cells, and that the cell membrane and membranefractions reassemble in the process. The yield of vesicles produced bysuch a method is much higher than that of naturally produced exosomes.

Artificial nanovesicles used in embodiments of the present invention maybe produced by serial extrusions of producer cells, typically mammaliancells, through micro- and/or nano-filters. The nanovesicles may beproduced by a method as described in US 2012/0177574, incorporatedherein by reference, see in particular paragraphs [0173]-[0197]. Thethus artificially produced nanovesicles retain the membrane structure ofthe producer cells, such as the lipid bilayer structure and membraneproteins including topology of the cell membrane surface molecules.Furthermore, the nanovesicles retain the same cytoplasmic components asthe producer cell.

A nanovesicle according to embodiments of the invention may have a sizeup to 500 nm, for example up to 300 nm, such as up to 250 nm or 200 nm.For example, the nanovesicles may have a size in the range of 100-200nm. However, in some embodiments the nanovesicles may be smaller than100 nm, for example at least 50 nm or at least 80 nm.

In embodiments of the invention, exosomes and other vesicles, e.g.microvesicles, naturally produced and released by cells may also containthe inhibitor molecule and optionally also express a targeting moleculeon its surface.

Exosomes, microvesicles and artificial nanovesicles have severalsimilarities but there are also important differences between thesevesicles. Exosomes are produced through a natural process, involvinginward budding of the cell membrane and are formed via multivesicularbodies. Exosomes are loaded with very specific materials, including RNA,which is not a random selection of cellular content. Furthermore,exosomes are very tight structures, which have been shown to bedifficult to load with external siRNAs (Wahlgren et al., Nucleic AcidsRes. 2012 Sep. 1; 40(17):e130). Microvesicles, on the other hand, seemin some experiment to contain much less RNA than exosomes.

Artificial nanovesicles, produced by serial extrusions of cells throughmicro- and nanofilters, do contain any protein on their surface that thecell does not have, and also contains a random selection of thecytoplasm. Therefore, overexpression of targeting surface molecule inthe cell membrane of an engineered cell will result in the presence ofthat molecule on the artificial nanovesicles. Similarly, a moleculeoverexpressed within the cell, will also be present within theartificial nanovesicle in high concentrations. Most importantly, theyield of artificial nanovesicles is expected to be significantly higherthan the yield of exosomes or other extracellular vesicles naturallyreleased by a cell.

In embodiments of the invention, the inhibitor molecule contained withinthe nanovesicles may be a nucleic acid, typically an RNAi molecule suchas shRNA, siRNA or miRNA, and typically shRNA or siRNA.

RNA interference, referred to as RNAi, is a gene regulating mechanismoccurring in many eukaryotic cells. RNAi is mediated by the Dicerenzyme, which cleaves a long double-stranded RNA molecule (dsRNA) intoshorter stands of double stranded RNA, small interfering RNA (siRNA) ThesiRNA strands are separated into two single-stranded RNA molecules,ssRNAs, and the guide strand is incorporated into a complex called theRNA-induced silencing complex (RISC) and may silence gene expression bycomplementary binding to a mRNA molecule. The RNAi mechanism can beutilized for selective silencing of a gene of interest by geneticallyengineered expression in a cell of a small hairpin RNA molecule (shRNA),containing a nucleotide sequence designed to silence a particular mRNA,and which is subsequently cleaved by Dicer into siRNA.

As used herein, “RNAi molecule” typically refers to any RNA moleculeinvolved in an RNAi process, including for example dsRNA, short hairpinRNA (shRNA, which may be used as a starting material instead of thedsRNA), miRNA, siRNA, and ssRNA.

The use of inhibitor oligonucleotides, and RNAi in particular, has manyadvantages over small molecule drugs. Small molecules exhibit problemswith solubility, which render them impossible to formulate for in vivodelivery. Once formulated they may also exhibit problems withabsorption, requiring delivery via intravenous, rather than an oral,route. The small molecule may also be bound by serum proteins orhindered from entering e.g. a tumor because of too high interstitialpressure, resulting in a poor distribution. Small molecules may also bedistributed systemically, which results in a lower intra-tumoralconcentration. Small molecules may also be metabolized, leading todecreased stability and the possible production of toxic bi-products.Decreased steady-state levels can also be the result of excretion.Finally, small molecules may have multiple targets and exhibit toxicityin a living multicellular organism. Oligonucleotides, on the other hand,are rapidly degraded into non-toxic residues.

In embodiments of the invention, the inhibitor molecule may be an RNAimolecule that can achieve inhibition of a member of the Myc family. Insuch embodiments, the inhibitor molecule may comprise an RNA sequenceselected from among SEQ ID NO: 93-124, for example selected from SEQ IDNO: 93-102 (RNA targeting c-myc), SEQ ID NO: 103-111 (targeting L-myc),or SEQ ID NO: 112-124 (targeting N-myc).

In embodiments of the invention the inhibitor molecule may be an RNAimolecule that can achieve inhibition of a member of the E2F family, forexample E2f1, E2f2 or E2f3. Such inhibitor molecules may comprise an RNAsequence selected from SEQ ID NO: 125-159, for example SEQ ID NO:125-140 (targeting E2f1), SEQ ID NO: 141-148 (targeting E2f2), or SEQ IDNO: 149-159 (targeting E2f3).

In yet other embodiments of the invention the inhibitor molecule may bean RNAi molecule that can achieve inhibition of a member of the Rasfamily, for example Hras, Kras or Nras. Such inhibitor molecules maycomprise an RNA sequence selected from SEQ ID NO: 160-184, for exampleselected from SEQ ID NO: 160-171 (targeting Hras), SEQ ID NO: 172-177(targeting Kras) or SEQ ID NO: 178-184 (targeting Nras).

In order to produce nanovesicles having the desired content of inhibitormolecules, in particular RNAi molecules, such as shRNA or siRNA, a DNAsequence coding for the inhibitor molecule may be inserted into theproducer cell using conventional genetic engineering techniques, e.g.using a viral vector. Vectors suitable for obtaining e.g. shRNAexpression in animal, e.g. mammalian, cells are known to a personskilled in the art, and may include vectors based on adeno-associatedviruses, adenoviruses or retroviruses/lentiviruses.

Hence, a DNA sequence encoding the inhibitor molecule of interest may beintroduced into the producer cells, which may be maintained underconditions allowing the expression of the DNA sequence, i.e. productionof the inhibitor molecule within the producer cells. By introduction ofa DNA sequence encoding the inhibitor molecule, the inhibitor moleculemay be expressed at high quantities within the producer cell andlocalized to the cytosol. For example, shRNA encoded by a DNA sequenceis translocated by the cellular machinery to the cytosol, where it maybe processed into siRNA. When the inhibitor molecule is expressed andpresent within cytosolic compartment of the producer cell, the inhibitormolecule will also be present in nanovesicles produced from the cells,since the nanovesicles have the same cytosolic composition as theproducer cell. The nanovesicles produced from cells expressing a shRNAwill thus contain, for example, shRNA and/or siRNA originating from theshRNA, typically siRNA.

Alternatively, an inhibitor molecule, such as an oligonucleotide, can beloaded directly into isolated nanovesicles.

In embodiments of the invention, the inhibitor molecule is an RNAimolecule, typically shRNA or siRNA, for inhibition or repression of ahuman MYC gene or gene product.

To express an RNAi molecule such as shRNA against human c-Myc, a DNAsequence may be introduced in the producer cell comprising a sequenceselected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID. NO: 8, and SEQ ID NO:9; alternatively, a DNA sequence may be introduced which is selectedfrom the group consisting of SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO:187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, and SEQ IDNO: 196.

To express an RNAi molecule such as shRNA against human L-Myc, a DNAsequence may be introduced in the producer cell comprising a sequenceselected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, and SEQ ID NO: 19. Alternatively, a DNA sequence may beintroduced which is selected from the group consisting of SEQ ID NO:197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, and SEQ ID NO: 205.

To express an RNAi molecule such as shRNA or siRNA against human N-Myc,a DNA sequence may be introduced in the producer cell comprising asequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ IDNO: 32. Alternatively, a DNA sequence may be introduced which isselected from the group consisting of SEQ ID NO: 206, SEQ ID NO: 207,SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ IDNO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216,SEQ ID NO: 217, and SEQ ID NO: 218.

Lentiviral plasmids containing the DNA sequences of SEQ ID NO: 185-218are commercially available from Sigma Aldrich (USA).

In embodiments of the invention, the inhibitor molecule is an RNAimolecule, typically shRNA or siRNA, for inhibition or repression of ahuman E2F gene or gene product. To express an RNAi molecule such asshRNA against human E2f1, a DNA sequence may be introduced in theproducer cell comprising a sequence selected from SEQ ID NO: 33-48. Toexpress an RNAi molecule such as shRNA against human E2f2, a DNAsequence may be introduced in the producer cell comprising a sequenceselected from SEQ ID NO: 49-56. To express an RNAi molecule such asshRNA against human E2f3, a DNA sequence may be introduced in theproducer cell comprising a sequence selected from SEQ ID NO: 57-67.

In embodiments of the invention, the inhibitor molecule is an RNAimolecule, typically shRNA or siRNA, for inhibition or repression of ahuman RAS gene or gene product. To express an RNAi molecule such asshRNA against human Hras, a DNA sequence may be introduced in theproducer cell comprising a sequence selected from SEQ ID NO: 68-79. Toexpress an RNAi molecule such as shRNA against human Kras, a DNAsequence may be introduced in the producer cell comprising a sequenceselected from SEQ ID NO: 80-85. To express an RNAi molecule such asshRNA against human Nras, a DNA sequence may be introduced in theproducer cell comprising a sequence selected from SEQ ID NO: 86-92.

The DNA sequence coding for the inhibitor molecule is typically underthe control of a promoter. The promoter may be a constitutive promoteror an inducible promoter.

In embodiments of the invention, for example where the producer cellsare human cells dependent of a functional human c-Myc protein for theirown proliferation, it may be preferable to use an inducible promoter, sothat expression of the inhibitor molecule can be initiated at a suitablepoint in time, such as when a desired number of producer cells or adesirable producer cell density has been achieved. Any suitableconventional promoter may be used, for example a doxycyclin-induciblepromoter.

In other embodiments, the producer cells may be independent of thefunction of the target gene which the inhibitor molecule is intended toinhibit or repress. In such embodiments, the promoter may be aconstitutive promoter or an inducible promoter. As used herein “targetgene” refers to a gene to be inhibited or repressed by the inhibitormolecule, or while gene product is to be inhibited or repressed by theinhibitor molecule.

For example, non-human animal cells or bacteria may be used as producercells for the production of nanovesicles containing an inhibitormolecule directed to a human gene or gene product. For instance, mousecells may be used for production of an RNAi molecule (and nanovesiclescontaining the RNAi molecule) against a human gene, e.g. a member of thehuman MYC gene family, such as human c-Myc, at least where the murineand human genes do not have too high sequence similarity. Alternatively,producer cells of the same species as the target gene may be used, whichhas been modified to be independent on the target gene. As an example,human cells to be used for the production of an RNAi molecule against afirst member of the MYC family may be modified to overexpress anothermember of the MYC gene family, so that it is made independent on thefunctionality of the first MYC member. In particular, human cells may begenetically modified using standard techniques to overexpress N-myc,which is normally expressed only at low levels, which may functionallyreplace c-Myc and render the cells independent of c-Myc. Hence,expression of an RNAi molecule against human c-MYC within the sameproducer cells will not disrupt cell growth and proliferation.Alternatively, the native c-Myc of a human cell may be geneticallymodified, e.g. by introduction of one or more point mutations so as toavoid complementarity with an RNAi molecule directed against human c-Mycand thus avoid inhibition of the c-Myc of the producer cell.

In embodiments of the invention, nanovesicles may be produced from cellsas described above, without insertion of a DNA sequence encoding theinhibitor molecule. In such embodiments, the inhibitor molecule may beintroduced (“loaded”) directly into the isolated nanovesicles usingelectroporation or the like. For example, an RNAi molecule such as siRNAmay be inserted directly into isolated nanovesicles by electroporation.Previous studies suggest that that this approach may be efficient fordelivery into exosomes (Alvarez-Erviti, L., et al., Delivery of siRNA tothe mouse brain by systemic injection of targeted exosomes. NatBiotechnol, 2011. 29(4): p. 341-5;).

The cells used as producer cells in the present invention may be anyeukaryotic cell that is capable of forming nanovesicles and which issusceptible of expressing an inhibitor molecule. Typically, the cellsmay be animal cell, in particular mammalian cells, including murine andhuman cells. Further, the cells used as producer cells may originatefrom a cell line suitable for in vitro proliferation, modification andexpression of the inhibitor molecule, and production of thenanovesicles. The cells may be stem cells. Examples of suitable cellsinclude human embryonic kidney (HEK, e.g. HEK 293) cells, mesenchymalstem cells, fibroblasts (e.g. NIH 3T3 mouse fibroblasts), or any othersuitable eukaryotic cells. Mesenchymal stem cells may be produced eitherfrom other cells, such as embryonic cells, or by autologous means (thusfrom the patient for which the therapy is intended)

In order to achieve selective delivery of the nanovesicles to a cancercell, the nanovesicles may have a targeting molecule present on theouter surface of its membrane. Preferably, the targeting molecule may bea molecule, e.g. a ligand, which selectively binds to another molecule,e.g. a ligand receptor, that is specific to the surface of cancer cellsor overrepresented (overexpressed) on the surface of cancer cells.Examples of suitable targeting molecules include epidermal growth factor(EGF), which binds to the EGF receptor (EGFR), andmelanocyte-stimulating hormone (MSH), which binds to the MSH receptor(MSHR; also called melanocortin 1 receptor, MC1R). EGFR is overexpressedon a majority of epithelial cancer cell types, whereas MC1R is expressedon melanoma and cells of the melanocyte lineage. Recently, it was shownthat exosomes that overexpress EGF-like peptides, using thetransmembrane domain of platelet-derived growth factor, can target toEGFR expressing tumors in vivo in mouse (Ohno, S. I., et al.,Systemically Injected Exosomes Targeted to EGFR Deliver AntitumorMicroRNA to Breast Cancer Cells. Mol Ther, 2012). Targeting moleculescan also include natural ligands or even engineered antibody fragmentspossessing the ability to bind cancer-specific surface antigens.

To introduce the targeting molecule into the nanovesicles, a DNAsequence comprising a DNA sequence, such as a gene, encoding thetargeting molecule may be introduced using conventional cloningtechniques, including any sequences such as a promoter etc. necessary toachieve expression of the targeting molecule within the cell. Thetargeting molecule may be a molecule that locates naturally to the outersurface of the cell membrane. Alternatively, the targeting molecule maybe a fusion protein with a transmembrane protein, such as a GPI-anchoredprotein or the transmembrane/cytosolic domain of ICAM-1 (T/C ICAM-1).

When the targeting molecule is localized to the cell membrane, thetargeting molecule will also form part of the nanovesicle membrane afterisolation of nanovesicles as described above. Genetic modification of aproducer cell to express a targeting molecule on the surface of the cellmembrane may be carried out before the step of introducing a DNAsequence encoding the inhibitor molecule into the producer cell.Alternatively, in embodiments of the invention, the DNA sequenceencoding the targeting molecule and the DNA sequence encoding theinhibitor molecule may be introduced into the producer cellsimultaneously. Introduction of a targeting molecule on the surface ofthe producer cell and hence also on its naturally occurringextracellular vesicles, or the nanovesicles produced by serial extrusionthrough membranes, may be performed also in embodiments where theproducer cells do not express the inhibitor molecules, and thenanovesicles are isolated and subsequently used for direct introductionof an inhibitor molecule e.g. via electroporation.

The present invention offers a means to develop successful therapiesagainst numerous diseases, including cancers. Nanovesicles according toembodiments of the invention may be administered to a patient sufferingfrom a cancer caused at least partly by the oncogene or proto-oncogeneagainst which the inhibitor molecule is directed. The nanovesicles maybe administered via intravenous injection or directly into tumors, orinto tumor-containing organs or parts of the body afflicted by tumors,optionally as part of a pharmaceutical composition.

A pharmaceutical composition may comprise the nanovesicles and typicallyalso a pharmaceutically acceptable carrier. Examples of pharmaceuticalcompositions suitable for injection include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. Typically the compositionis sterile. Further, the composition may be fluid to the extent thateasy syringeability exists. Preferably, the composition is stable underthe conditions of manufacture and storage, and it is preferablypreserved against the contaminating action of microorganisms, such asbacteria and fungi.

Administration of a pharmaceutical composition according to embodimentsof the invention may be effected via any suitable route, for exampleintravenous, intradermal, intramuscular, into brain, into spinal fluid,subcutaneous, directly into tumors or tumor infested tissues, or limbs,or intraocular, or intranasal, or by inhalation, or oral and/orintraperitoneal.

The nanovesicles may be internalized by cancer cells in a way similar toexosomes and other vesicles, and hence deliver their cargo of inhibitormolecule to the interior of a cancer cell, where the inhibitor moleculecan perform its inhibitor function, e.g. via RNAi.

Additionally or alternatively, producer cells according to embodimentsof the invention may be administered directly to a patient, for exampledirectly into a tumor, without prior isolation of nanovesicles. In suchcases, the cells preferably express a targeting molecule on the cellsurface, so that they are localized in close vicinity to cancer cellsafter administration. Administration may be effected via any suitableroute, for example intravenous, intradermal, intramuscular, into brain,into spinal fluid, subcutaneous, directly into tumors or tumor infestedtissues, or limbs, or intraocular, or intranasal, or by inhalation, ororal and/or intraperitoneal. Further, in such cases the DNA sequenceencoding the molecule inhibitor is preferably under the control of aninducible promoter, such as a promoter inducible by doxycyclin. Afteradministration of the cells, expression of the inhibitor molecule may beinduced by administration of doxycyclin to the patient by any suitableroute, e.g. orally or intravenously. Upon expression of an RNAimolecule, the cells will be stressed and as a result release highamounts of exosomes and other vesicles during a short period of time,which will lead to high concentration of RNAi containing vesicles in thevicinity of the tumor cells, which then through natural means will takeup the vesicles, and the RNAi molecule will have its therapeutic effectinside of the tumor cell by inhibiting crucial proto-oncogenes such asc-Myc or other transcription factors. High and/or prolonged expressionof the inhibitor molecule in the injected cell will most likely induceapoptosis, which can result in further release of the molecule inhibitorfrom the cell via apoptotic bodies, which also can deliver their cargoto recipient tumor cells. As a result, the RNAi molecule will beencapsulated in naturally occurring nano- and microvesicles in the closevicinity of tumor cells, and will subsequently enter the cancer cellsand elicit RNAi. The encapsulation of the RNAi molecule into vesicles isimportant, since the RNAi molecules would be naturally degraded ifpresent as free molecules in the interstitial tissue (extracellularly).

The present invention offers a therapeutic tool that could potentiallybe used in the treatment of numerous cancerous diseases. The c-Myc genemay be overexpressed in as much as 80% of all cancers (Nilsson &Cleveland Oncogene. 2003 Dec. 8; 22(56):9007-21).

One example of a cancer that could be treated using cells ornanovesicles according to embodiments of the present invention ismelanoma. It has previously been shown that c-Myc inhibition can beefficient in causing senescence in a majority of malignant humanmelanoma cell lines (Wang et al., 2008). Further, Myc inhibition bysystemic expression of a dominant-interfering Myc mutant in a mousemodel of Ras-induced lung adenocarcinoma, triggers rapid regression ofincipient and established lung tumors, confirming that targeting Myc maybe efficient in treatment of malignancy (Soucek, L., et al., ModellingMyc inhibition as a cancer therapy. Nature, 2008. 455(7213): p. 679-83).Together, these previous studies suggest that many malignanciesincluding melanoma are dependent on c-Myc.

A nanovesicle according to embodiments of the invention that a) targetsmalignant melanoma cells, and b) contains an RNAi molecule thatdown-regulates a downstream oncogene or a crucial transcription factorfor the melanoma cells, may therefore be possible to utilize inmetastatic uveal melanoma, as well as metastatic skin melanomas,regardless of oncogene mutation status. The c-Myc and E2f1 genes arebelieved to be very suitable target genes for such therapy, as they hasbeen proven to be crucial in melanoma in general. Lastly, amelanoma-targeting cell or nanovesicle could possibly also be used aslocal therapy in the eye in uveal melanoma without metastasis, or asadjuvant therapy together with local removal of tumor, possibly avoidingenucleation.

c-Myc has also been associated with carcinoma of the cervix, colon,breast, lung and stomach, and the invention may thus also be useful intherapy of such malignancies.

Hence, in addition to melanoma, cells and/or nanovesicles targeting forexample EGFR positive malignancies and containing an inhibitor to c-Myc,can be a putative therapeutic approach for many malignant diseases.

EXAMPLES Example 1 Introducing Targeting Molecule on the Surface ofHuman Producer Cells

Firstly, pCMV-zeo vectors expressing EGF or MSH in frame with either aGPI-anchor protein or I-CAM1 are created and transfected into HEK 293cells or mesenchymal stem cells. The transfected cells are treated withzeocin and resistant cells are sorted based on surface expression of EGFor MSH using magnetic beads or a cell sorter. Maintenance of thetransgenic expression may be routinely monitored with flow cytometry.

The cells expressing EGF or MSH fusion proteins may be further used forproduction of nanovesicles, optionally with expression of an inhibitormolecule.

Example 2 Production of Nanovesicles Using Human Cells Modified to beIndependent of c-Myc

A. Modification of Human Cells to Become Independent on c-Myc

A mammalian expression vector containing a murine retrovirus receptor(mCAT-1) and an N-Myc:GFP fusion separated by an IRES (to enablecap-independent translation) is transfected into HEK 293T cells ormesenchymal stem cells. The cell line used may be equipped with anecotropic receptor for murine-specific envelope-pseudotyped lentivirusto increase personnel safety. Further, by using a pcDNA-neo vector forthe cloning it is possible to also express a neomycin resistancecassette. Alternatively, instead of an N-Myc gene the expression vectormay contain an L-myc gene, if the inhibitor is directed to c-Myc.

The vector is cloned into human cells using the Amaxa nucleofector, orany other suitable methodology. Successfully transduced cells areselected with neomycin and FACS sort for GFP positive cells.

B. Introduction of a DNA Sequence Encoding an Inhibitor to a Human MycProtein into the Nanovesicle-Producing Cells

A lentiviral plasmid obtained from Sigma Aldrich, USA (MISSIONTRCpLKO-puro clones) having a sequence selected from the groupconsisting of SEQ ID NO: 185-196 is transfected into HEK 293T cells.When transfected into cells HIV-1 based transcripts are generated whichhave been made replication-incompetent and self-inactivating by adeletion in the 3′-long terminal repeat. Further, the lentiviral vectoris co-transfected with pCMV-dR8.2 dvpr and pHCMV-Eco (both from theAddgene plasmid depository), the former containing a minimal vectorexpressing HIV-GAG and the latter an eco-tropic envelope. The HEK 293Tcells will thus produce lentivirus that are replication-incompetent andcan only infect murine cells or cells expressing the murine retrovirusreceptor (mCAT1) as in Example 2A. Next, the HEK 293T cells are culturedand virus-containing cell culture supernatant from the transfected cellsis used to transduce mCAT1-expressing human cells, followed by selectionwith puromycin (encoded by lentivirus plasmid). Since themCAT1-expressing human cells depend on the N-Myc gene and not on thehuman target gene, e.g. c-Myc, their growth or viability may beunaffected by the expression of an inhibitor molecule directed against ahuman target gene, e.g. human c-Myc.

C. Isolation of Nanovesicles

Isolation of nanovesicles, and or naturally occurring extracellularvesicles, can be performed by known procedures, which includescentrifugation steps. For example, artificial nanovesicles may beproduced as described in US 2012/0177574. Artificial high-densityvesicles, emerging from nuclear membranes can be removed by a briefcentrifugation steps. Nanovesicles expressing the molecule that targetsfor example the EGF receptor may be purified either by positive ornegative selection, using either advanced FACS sorting protocols,magnetic methods or any other method, using antibody technology.

Example 3 Production of Nanovesicles Using Human Cells (InducibleExpression)

A. Introduction of a DNA Sequence Encoding an Inhibitor Against a MycProtein (Inducible Expression) into Human Cells

A tetracycline (doxycycline) or IPTG-inducible expression system is usedfor cloning the DNA sequence of the desired inhibitor molecule (here aDNA sequence according to any one of SEQ ID NO: 185-218. For example,the pLKO-TetON-puro system (Addgene) or the pLKO_IPTG_3×LacO system(Sigma) may be used.

B. Isolation of Nanovesicles (Optional)

Isolation of nanovesicles, and or naturally occurring extracellularvesicles, can be performed by known procedures, which includescentrifugation steps. For example, artificial nanovesicles may beproduced as described in US 2012/0177574. Artificial high-densityvesicles, emerging from nuclear membranes can be removed by a briefcentrifugation steps. Nanovesicles expressing the molecule that targetsfor example the EGF receptor may be purified either by positive ornegative selection, using either advanced FACS sorting protocols,magnetic methods or any other method, using antibody technology.

Alternatively, instead of isolating nanovesicles, the inhibitor moleculemay be recovered from the cell culture and used later for directintroduction into nanovesicles e.g. using electroporation. In suchembodiments, the inhibitor molecules may be introduced into artificiallyproduced nanovesicles as described above.

Alternatively, in stead of using producing artificial nanovesicles bysequential extrusion, extracellular vesicles that are naturally releasedfrom the engineered producer cell, for example exosomes, microvesiclesor apoptotic bodies, may be isolated from the cell culture. Such naturalextracellular vesicles will express both the targeting molecule on itssurface, as well as contain the inhibitor molecule.

Example 4 Production of Nanovesicles Using Murine Cells

A. Introduction of DNA Sequence Encoding a Myc Inhibitor into MurineCells

A lentiviral plasmid obtained from Sigma Aldrich, USA (MISSIONTRCpLKO-puro clones) having a sequence selected from the groupconsisting of SEQ ID NO: 185-218 is transfected into HEK 293T cells.When transfected into cells HIV-1 based transcripts are generated whichhave been made replication-incompetent by a deletion in the 3′-longterminal repeat. Further, the lentiviral vector is co-transfected withpCMV-dR8.2 dvpr and pHCMV-Eco (both from the Addgene plasmiddepository), the former containing a minimal vector expressing HIV-GAGand the latter an eco-tropic envelope. The HEK 293T cells will thusproduce lentivirus that are replication-incompetent and can only infectmurine cells. Next, the HEK 293T cells are cultured and virus-containingcell culture supernatant from the transfected cells is used to transduceNIH 3T3 mouse fibroblasts, followed by selection with puromycin (encodedby lentivirus plasmid). Since the mouse fibroblasts depend on theirnative mouse gene and not on the human target gene, e.g. c-Myc, theirgrowth or viability may be unaffected by the expression of an inhibitormolecule directed against a human target gene, e.g. human c-Myc.

B. Isolation of Nanovesicles

Isolation of nanovesicles, and/or naturally occurring extracellularvesicles, can be performed by known procedures, which includescentrifugation steps. For example, artificial nanovesicles may beproduced as described in US 2012/0177574. Artificial High-densityvesicles, emerging from nuclear membranes can be removed by a briefcentrifugation steps. Nanovesicles expressing the molecule that targetsfor example the EGF receptor may be purified either by positive ornegative selection, using either advanced FACS sorting protocols,magnetic methods or any other method, using antibody technology.

The invention claimed is:
 1. A method of producing nanovesiclescomprising an inhibitor oligonucleotide to an oncogene or aproto-oncogene or the gene product thereof, said method comprising (a)transfecting or transducing a mammalian cell with a construct comprisingL-Myc genes; (b) maintaining the cell under conditions allowing the cellto overexpress L-Myc; c) introducing into the cell a DNA sequenceencoding an oligonucleotide capable of inhibiting a human oncogenic orproto-oncogeneic transcription factor wherein said inhibitor does notinhibit L-Myc; and (d) obtaining nanovesicles containing said inhibitoroligonucleotide from said cell.
 2. A method according to claim 1,wherein said inhibitor oligonucleotide is an inhibitor to a human MYCgene or gene product, preferably the c-Myc gene or gene product.
 3. Amethod according to claim 1, wherein said oligonucleotide is RNA,preferably an RNAi molecule.
 4. A method according to claim 3, whereinsaid DNA sequence encoding an oligonucleotide capable of inhibiting ahuman oncogenic or proto-oncogenic transcription factor is a DNAsequence selected from the group consisting of SEQ ID NO: 1-9.
 5. Amethod according to claim 1, wherein said cell expresses a targetingmolecule on the cell surface.
 6. A method according to claim 1, whereinthe method comprises, prior to step (c) or simultaneously with step (c),introducing a DNA sequence encoding a targeting molecule to be expressedon the cell surface.
 7. A method according to claim 5, wherein saidtargeting molecule is a ligand specifically binding to a surface proteinthat is overexpressed on cancer cells.
 8. A method according to claim 7,wherein said targeting molecule is epidermal growth factor (EGF),melanocyte-stimulating hormone (MSH) or a variable region of an antibodydirected against a surface protein that is overexpressed on cancercells, or a fab-fragment of said antibody.
 9. A method according toclaim 1, wherein the cell is a human cell.
 10. A method according toclaim 1, wherein step (c) comprises introducing a genetic constructcomprising said DNA sequence coding for an oligonucleotide capable ofinhibiting human c-Myc, operatively linked to an inducible promoter, andinducing expression by activation of said inducible promoter.
 11. Amethod according to claim 1, performed in vitro or ex vivo.
 12. A methodaccording to claim 3, wherein said DNA sequence encoding anoligonucleotide capable of inhibiting a human oncogenic orproto-oncogenic transcription factor is a DNA sequence selected from thegroup consisting of SEQ ID NO: 185-196.